Following myocardial ischemia and reperfusion (I-R), bioenergetic dysfunction and loss of calcium homeostasis contribute to poor functional recovery. Recent findings highlight three underlying phenomena, including the role of reactive nitrogen species (RNS), mitochondrial perturbations, and MAP kinase signaling. Although these mechanisms are thought to be related, this has not been addressed. Developments in mitochondrial proteomics and new insights into nitric oxide (NO) interactions with mitochondria allow integration of these mechanisms, and serve as the theme of this proposal. Understanding the order and mechanism of events upstream of mitochondrial perturbations is clinically important, since many pharmacologic approaches to I-R injury are directed at modulating RNS levels, mitochondria, and MAP kinase signaling. Also, the therapeutic roles of RNS in I-R have been difficult to address, since RNS exhibit both beneficial and damaging effects in I-R. Building upon data presented herein it is proposed that recent insights into the interactions of RNS and calcium with mitochondria during I-R can reveal underlying pathological mechanisms. This will be investigated using assessment of mitochondrial function and a proteomics approach. Preliminary data indicate that dependent upon exposure of mitochondria to Ca2+ or RNS, different targets are revealed, and determine the outcome of injury. In addition, a novel role for MAP kinases in I-R-induced Ca2+ elevation is proposed. The focus of the proposal are two mitochondrial parameters that alter in response to both I-R and RNS exposure: These are the inhibition of respiratory complex I activity, and an elevation in mitochondrial H+ leak. It is hypothesized that RNS, calcium, and MAP kinases are responsible for the inhibition of mitochondrial respiratory complex I and increased H+ leak in cardiac I-R. This hypothesis will be tested by pursuit of the following aims: 1. Apply mitochondrial proteomics to determine the mechanism of complex I inhibition in ischemia-reperfusion, and the effects of endogenous and exogenous NO. 2. Determine the mechanism of increased H+ leak in cardiac I-R and its regulation by RNS. 3. Investigate the role of MAP kinases in eliciting mitochondrial dysfunction in cardiac I-R. Realization of these aims will greatly enhance understanding of the basic mechanisms underlying mitochondrial perturbations in I-R leading to novel therapies.